Extract of adlay bran and uses thereof

ABSTRACT

The present invention provides a composition comprising extract of adlay bran, wherein the adlay bran extract comprises C1 to C7 alcohol extract of adlay bran (A) and carbon dioxide supercritical fluid extract of adlay bran (B). Preferably, the extract of adlay bran has effects in treating a skin and/or subcutaneous tissue disease and in lowering interleukin (IL)-1α, interleukin-1β, interleukin-6, tumor necrosis factor (TNF)-α, interleukin-8, prostaglandin-2 (PGE2) and/or C-Reactive Protein (CRP).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to extract of adlay bran and uses thereof; moreparticularly, the uses in treating a skin and/or subcutaneous tissuedisease and lowering interleukin (IL)-1α, interleukin-1β, interleukin-6,tumor necrosis factor (TNF)-α, interleukin-8, prostaglandin-2 (PGE2)and/or C-Reactive Protein (CRP).

2. Description of the Related Art

The skin is the largest organ in the human body and is the main barrierbetween the body and the environment, and is the first line of defensefrom pathogens and physical and chemical stresses or stimulations. Theskin not only provides the physical and chemical protection, but also isan immune organ that can effectively cause passive and active immuneresponse, thereby protecting the human body.

From outside to inside, the skin has a three-layer structure composed ofthe epidermis, the dermis and the subcutaneous tissue. The outmost layerepidermis is responsible for protection functions; the dermis layerinside the epidermis layer is the most important part of the skin, andis responsible for supporting the epidermis and interlaces with variousfibers to form a support net, and blood vessels, nerves, sebaceousglands, sweat glands and hair follicles are distributed in the dermislayer; the innermost layer of the skin is the subcutaneous tissue, whichin general refers to the deep layer of the dermis of vertebrates, and innarrow sense refers to the adipose connective tissues between the dermisand skeleton and muscles below the dermis. The subcutaneous tissuemainly formed by fat cells has no obvious boundary with the dermis, andis responsible for blocking and absorbing vibration and providing asource of energy. The subcutaneous tissue is a main site for fatmetabolism.

The skin and/or the subcutaneous tissue may have various diseases due tosome congenital or acquired factors, resulting in discomfort ofpatients, and changes in appearance cause psychological burdens onpatients. However, the currently adopted skin and/or subcutaneous tissueagents are steroids or acidic agents, and great side effects may becaused by improper use, so use of the existing agents are not anappropriate treatment.

In the skin and/or subcutaneous tissue diseases, damages caused byradiation therapy is one of the important types, and the radiationtherapy always induces side effects such as radiation dermatitis,fatigue, radiation pneumonia and lymphedema (McCormick et al., 1989, IntJ Radiat Oncol Biol Phys., 17:1299-1302; Lingos et al., 1991, Int. J.Radiat. Oncol. Biol. Phys., 21:355-360; Taylor et al., 1995, Int J.Radiat. Oncol. Biol. Phys., 31:753-764; Gorodetsky et al., 1999, Int. J.Radiat. Oncol. Biol. Phys., 45:893-900; Erickson et al., 2001, J. Natl.Cancer Inst, 93:96-111). With breast cancer as an example, about 50% to60% breast cancer patients need radiation therapy after operation. Afteroperation resection and systemic chemotherapy, the side effects such ashair loss, neutropenia, nausea and vomiting always occur in thepatients. As a result, the patients may evade or refuse the treatmentbecause of fear. The medical community has made some improvements in thetreatment equipment and technology to reduce the side effects of theradiation therapy. As for skin erythema and pigmentation caused byradiation and some serious side effects such as ulcer in a minority ofthe patients, the medical community always tries to use some radiationprotection agents such as Amifostine (Yuhas et al., 1980, Cancer Clin.Trials., 3:211-216). However, the agents are almost not used in theactual medical practice due to accompanying strong side effects,non-obvious effect, or high price. An agent that has the effect ofradiation protection or radiation effect strengthening and can reducethe dose of radiation is still needed, so as to bring benefit for cancerpatients receiving the radiation therapy.

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) seeds, also calledJob's tears, are a component of traditional Chinese medicine (TCM) andhave long been used as an anti-inflammatory agent to treat warts,chapped skin, rheumatism, and neuralgia (Li, S. C. Pen-t'sao kangmu(Systematic Pharmacopoeia); China, 1596). A recent study showed thatdehulled adlay (DA) modulated the microbiota in the intestinal tract ofrats (Chiang, W.; Cheng, C.; Chiang, M.; Chung, K. T. J. Agric. FoodChem. 2000, 48, 829-832.). Also, the anti-inflammatory and antioxidativeeffects of adlay were elucidated in vitro (Lee, M. Y.; Tsai, S. H.; Kuo,Y. H.; Chiang, W. Food Sci. Biotechnol. 2008, 17, 1265-1271; Kuo, C. C.;Shih, M. C.; Kuo, Y. H.; Chiang, W. J. Agric. Food Chem. 2001, 49,1564-1570). Contents of various potent compounds in adlay seeds fromdifferent origins were quantified (Wu, T. T.; Charles, A. L.; Huang, T.C. Food Chem. 2007, 104, 1509-1515). Several phenolic antioxidants wereisolated from adlay seeds, and bioactive components in adlay seeds werefound to be stable during processing (Hsu, H. Y.; Lin, B. F.; Lin, J.Y.; Kuo, C. C.; Chiang, W. J. Agric. Food Chem. 2003, 51, 3763-3769).Lignans and phenolic compounds were isolated from adlay hull (AH) in anassay-guided isolation (Kuo, C. C.; Shih, M. C.; Kuo, Y. H.; Chiang, W.J. Agric. Food Chem. 2001, 49, 1564-1570). Flavanone and severalphenolic acids were isolated from anti-inflammatory fractions of adlayseeds (Huang, D. W.; Kuo, Y. H.; Lin, F. Y.; Lin, Y. L.; Chiang, W. J.Agric. Food Chem. 2009, 57, 2259-2266; Huang, D. W., Chung, C. P.; Kuo,Y. H.; Lin, Y. L.; Chiang, W. J. Agric. Food Chem. 2009, 57,10651-10657; Chen, H. J.; Chung, C. P.; Chiang, W.; Lin, Y. L. FoodChem. 2011, 126, 1741-1748). Phenolic alcohol in the adlay testa (AT)was reported to possess antiallergic activity (Chen, H. J.; Shih, C. K.;Hsu, H. Y.; Chiang, W. J. Agric. Food Chem. 2010, 58, 2596-2601). Inaddition, DA and adlay bran (AB) were shown to retard carcinogenesisthrough an anti-inflammatory pathway (Shih, C. K.; Chiang, W.; Kuo, M.L. Food Chem. Toxicol. 2004, 42, 1339-1347; Li, S. C.; Chen, C. M.; Lin,S. H.; Chiang, W.; Shih, C. K. J. Sci. Food Agric. 2011, 91, 547-552),and ferulic acid was regarded as the active component in a furtherinvestigation (Chung, C. P.; Hsu, H. Y.; Huang, D. W.; Hsu, H. H.; Lin,J. T.; Shih, C. K.; Chiang, W. J. Agric. Food Chem. 2010, 58,7616-7623).

Although there are many uses of adlay seeds reported, variousapplications of extract of adlay seeds remain to be developed.

SUMMARY OF THE INVENTION

In the present invention, extract of adlay bran has effects in treatinga skin and/or subcutaneous tissue disease and in loweringinterleukin-1α, interleukin-1β, interleukin-6, tumor necrosis factor-α,interleukin-8, prostaglandin-2 and/or C-Reactive Protein. Inparticularly, the extract of adlay bran provides radiation protection orenhances radiation efficacy to reduce the dose of radiation and furtherto benefit cancer patients receiving the radiation therapy.

The present invention provides a composition comprising extract of adlaybran, wherein the extract of adlay bran comprises C1 to C7 alcoholextract of adlay bran (A) and carbon dioxide supercritical fluid extractof adlay bran (B), and the weight ratio of the C1 to C7 alcohol extractof adlay bran (A) to the carbon dioxide supercritical fluid extract ofadlay bran (B) is from about 3:1 to about 1:4.

The invention also provides a method for treating a skin and/orsubcutaneous tissue disease in a subject, which comprises administeringto said subject an effective amount of extract of adlay bran andoptionally a pharmaceutically acceptable carrier or excipient, whereinthe extract of adlay bran comprises C1 to C7 alcohol extract of adlaybran (A) and carbon dioxide supercritical fluid extract of adlay bran(B), and the weight ratio of the C1 to C7 alcohol extract of adlay bran(A) to the carbon dioxide supercritical fluid extract of adlay bran (B)is from about 3:1 to about 1:4.

The invention also provides use of extract of adlay bran in themanufacture of a medicament of the treatment of a skin and/orsubcutaneous tissue disease, wherein the extract of adlay bran comprisesC1 to C7 alcohol extract of adlay bran (A) and carbon dioxidesupercritical fluid extract of adlay bran (B), and the weight ratio ofthe C1 to C7 alcohol extract of adlay bran (A) to the carbon dioxidesupercritical fluid extract of adlay bran (B) is from about 3:1 to about1:4.

The invention further provides a method for lowering interleukin-1α,interleukin-1β, interleukin-6, tumor necrosis factor-α, interleukin-8,prostaglandin-2 and/or C-Reactive Protein in a subject, which comprisesadministering to said subject an effective amount of extract of adlaybran and optionally a pharmaceutically acceptable carrier or excipient,wherein the extract of adlay bran comprises C1 to C7 alcohol extract ofadlay bran (A) and carbon dioxide supercritical fluid extract of adlaybran (B), and the weight ratio of the C1 to C7 alcohol extract of adlaybran (A) to the carbon dioxide supercritical fluid extract of adlay bran(B) is from about 3:1 to about 1:4.

The invention also provides use of extract of adlay bran in themanufacture of a medicament of lowering interleukin-1α, interleukin-1β,interleukin-6, tumor necrosis factor-α, interleukin-8, prostaglandin-2and/or C-Reactive Protein, wherein the extract of adlay bran comprisesC1 to C7 alcohol extract of adlay bran (A) and carbon dioxidesupercritical fluid extract of adlay bran (B), and the weight ratio ofthe C1 to C7 alcohol extract of adlay bran (A) to the carbon dioxidesupercritical fluid extract of adlay bran (B) is from about 3:1 to about1:4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the HPLC spectrogram of the C1 to C7 alcohol extractof adlay bran (A) according to the invention.

FIG. 2 illustrates the HPLC spectrogram of the carbon dioxidesupercritical fluid extract of adlay bran (B) according to theinvention.

FIG. 3 illustrates the HPLC spectrogram of the extract of adlay branaccording to the invention with the 1:4 weight ratio of the C1 to C7alcohol extract of adlay bran (A) to the carbon dioxide supercriticalfluid extract of adlay bran (B).

FIG. 4 illustrates the tissue pathological slices showing effects of theadlay capsules at different feeding doses or the agent Sulfasil fortreating the skin injuries caused by clinical radiation therapy on theradiation therapy-caused mouse leg fur tissue damages. RT representsradiation therapy; sham represents without radiation therapy; andvehicle represents no carrier of adlay extract contained.

FIG. 5 illustrates the results of body weight changes of the tumoranimals receiving the radiation therapy and at the same time, are fedwith the adlay capsule of different concentrations or the agent Sulfasilfor treating the skin injuries caused by clinical radiation therapythrough a tube. RT represents radiation therapy; sham represents withoutradiation therapy; and vehicle represents no carrier of adlay extractcontained.

FIG. 6 illustrates the results of changes of the tumor volume of thetumor animals receiving the radiation therapy and at the same time, arefed with the adlay capsule of different concentrations or the agentSulfasil for treating the skin injuries caused by clinical radiationtherapy through a tube. RT represents radiation therapy; sham representswithout radiation therapy; and vehicle represents no carrier of adlayextract contained.

FIG. 7 illustrates the results of changes of serum IL-1α concentrationof the tumor animals receiving the radiation therapy and at the sametime, are fed with the adlay capsule of different concentrations or theagent Sulfasil for treating the skin injuries caused by clinicalradiation therapy through a tube. RT represents radiation therapy; shamrepresents without radiation therapy; and vehicle represents no carrierof adlay extract contained.

FIG. 8 illustrates the results of changes of serum IL-1β concentrationof the tumor animals receiving the radiation therapy and at the sametime, are fed with the adlay capsule of different concentrations or theagent Sulfasil for treating the skin injuries caused by clinicalradiation therapy through a tube. RT represents radiation therapy; shamrepresents without radiation therapy; and vehicle represents no carrierof adlay extract contained.

FIG. 9 illustrates the results of changes of serum IL-6 concentration ofthe tumor animals receiving the radiation therapy and at the same time,are fed with the adlay capsule of different concentrations or the agentSulfasil for treating the skin injuries caused by clinical radiationtherapy through a tube. RT represents radiation therapy; sham representswithout radiation therapy; and vehicle represents no carrier of adlayextract contained.

FIG. 10 illustrates the results of changes of serum TNF-α concentrationof the tumor animals receiving the radiation therapy and at the sametime, are fed with the adlay capsule of different concentrations or theagent Sulfasil for treating the skin injuries caused by clinicalradiation therapy through a tube. RT represents radiation therapy; shamrepresents without radiation therapy; and vehicle represents no carrierof adlay extract contained.

FIG. 11 illustrates the results of changes of serum IL-8 concentrationof the tumor animals receiving the radiation therapy and at the sametime, are fed with the adlay capsule of different concentrations or theagent Sulfasil for treating the skin injuries caused by clinicalradiation therapy through a tube. RT represents radiation therapy; shamrepresents without radiation therapy; and vehicle represents no carrierof adlay extract contained.

FIG. 12 illustrates the results of changes of serum PGE2 concentrationof the tumor animals receiving the radiation therapy and at the sametime, are fed with the adlay capsule of different concentrations or theagent Sulfasil for treating the skin injuries caused by clinicalradiation therapy through a tube. RT represents radiation therapy; shamrepresents without radiation therapy; and vehicle represents no carrierof adlay extract contained.

FIG. 13 illustrates the results of changes of serum IL-1α concentrationof the tumor animals receiving the radiation therapy and at the sametime, are fed with the adlay capsule of different concentrations or theagent Sulfasil for treating the skin injuries caused by clinicalradiation therapy through a tube. RT represents radiation therapy; shamrepresents without radiation therapy; and vehicle represents no carrierof adlay extract contained.

FIG. 14 illustrates the tissue pathological slices showing effects of 25different feeding doses/combinations on the radiation therapy-causedmouse leg fur tissue damages. The horizontal axis and the vertical axisrespectively represent the feeding dose/combination (mg/kg) of the C1 toC7 alcohol extract of adlay bran (A) and the carbon dioxidesupercritical fluid extract of adlay bran (B). Each figure respectivelyillustrates the tissue pathological slices of fur tissues of mice thatare fed with different doses through a tube in the process of radiationtherapy of tumor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a composition comprising extract of adlaybran, wherein the extract of adlay bran comprises C1 to C7 alcoholextract of adlay bran (A) and carbon dioxide supercritical fluid extractof adlay bran (B), and the weight ratio of the C1 to C7 alcohol extractof adlay bran (A) to the carbon dioxide supercritical fluid extract ofadlay bran (B) is from about 3:1 to about 1:4.

The present invention can be more readily understood by reference to thefollowing detailed description of various embodiments of the invention,the examples, and the chemical drawings and tables with their relevantdescriptions. It is to be understood that unless otherwise specificallyindicated by the claims, the invention is not limited to specificpreparation methods, carriers or formulations, or to particular modes offormulating the extract of the invention into products or compositionsintended for topical, oral or parenteral administration, because as oneof ordinary skill in the relevant arts is well aware, such things can,of course, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meaning:

Often, ranges are expressed herein as from “about” one particular valueand/or to “about” another particular value. When such a range isexpressed, an embodiment includes the range from the one particularvalue and/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the word “about,” it will beunderstood that the particular value forms another embodiment. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to and independently of the other endpoint.As used herein the term “about” refers to ±10%.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally comprising an agent” meansthat the agent may or may not exist.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, unless otherwiserequired by context, singular terms shall include the plural and pluralterms shall include the singular.

The term “subject” as used herein denotes any animal, preferably amammal, and more preferably a human. The examples of subjects includehumans, non-human primates, rodents, guinea pigs, rabbits, sheep, pigs,goats, cows, horses, dogs and cats.

The term “effective amount” of an active ingredient as provided hereinmeans a sufficient amount of the ingredient to provide the desiredregulation of a desired function, such as gene expression, proteinfunction, or the induction of a particular type of response. As will bepointed out below, the exact amount required will vary from subject tosubject, depending on the disease state, physical conditions, age, sex,species and weight of the subject, the specific identity and formulationof the composition, etc. Dosage regimens may be adjusted to induce theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. Thus, it isnot possible to specify an exact “effective amount.” However, anappropriate effective amount can be determined by one of ordinary skillin the art using only routine experimentation.

The term “treating” or “treatment” as used herein denotes reversing,alleviating, inhibiting the progress of or improving the disorder,disease or condition to which such term applies, or one or more symptomsof such disorder, disease or condition.

The term “carrier” or “excipient” as used herein refers to anysubstance, not itself a therapeutic agent, used as a carrier and/ordiluent and/or adjuvant, or vehicle for delivery of a therapeutic agentto a subject or added to a formulation to improve its handling orstorage properties or to permit or facilitate formation of a dose unitof the composition into a discrete article such as a capsule or tabletsuitable for oral administration. Suitable carriers or excipients arewell known to persons of ordinary skill in the art of manufacturingpharmaceutical formulations or food products. Carriers or excipients caninclude, by way of illustration and not limitation, buffers, diluents,disintegrants, binding agents, adhesives, wetting agents, polymers,lubricants, glidants, substances added to mask or counteract adisagreeable taste or odor, flavors, dyes, fragrances, and substancesadded to improve appearance of the composition. Acceptable carriers orexcipients include citrate buffer, phosphate buffer, acetate buffer,bicarbonate buffer, stearic acid, magnesium stearate, magnesium oxide,sodium and calcium salts of phosphoric and sulfuric acids, magnesiumcarbonate, talc, gelatin, acacia gum, sodium alginate, pectin, dextrin,mannitol, sorbitol, lactose, sucrose, starches, gelatin, cellulosicmaterials (such as cellulose esters of alkanoic acids and cellulosealkyl esters), low melting wax cocoa butter, amino acids, urea,alcohols, ascorbic acid, phospholipids, proteins (for example, serumalbumin), ethylenediamine tetraacetic acid (EDTA), dimethyl sulfoxide(DMSO), sodium chloride or other salts, liposomes, mannitol, sorbitol,glycerol or powder, polymers (such as polyvinyl-pyrrolidone, polyvinylalcohol, and polyethylene glycols), and other pharmaceuticallyacceptable materials. The carrier should not destroy the pharmacologicalactivity of the therapeutic agent and should be non-toxic whenadministered in doses sufficient to deliver a therapeutic amount of theagent.

The composition according to the invention comprises the extract ofadlay bran. The adlay bran according to the invention preferably isobtained from dehulled adlay seeds. The term “dehulled adlay seeds” asused herein refers to seeds of adlay without hulls, testas, coverings,shells, or pods. The manner of removing the hulls, coverings, shells orpods from the adlay seeds is well-known to artisans skilled in thisfield. In general, the dehulled adlay seeds comprises bran andendosperm, and the manner of obtaining the bran from the dehulled adlayseeds is well-known to artisans skilled in this field.

The adlay seeds referred to in this invention are not particularlylimited. Preferably, the adlay belongs to Gramineae family, Panicoideaesub-family, and Coix species, or Poales order, Poaceae family, and Coixspecies. More preferably, the adlay is Coix lachryma-jobi, Coixlachryma-jobi L., Coix lachryma-jobi L. var. ma-yuen Stapf, Coixagrestis Lour., Coix arundinacea Lam., Coix exaltata Jacq., or Coixlacryma L.

The extract of adlay bran according to the invention comprises the C1 toC7 alcohol extract of adlay bran (A) and the carbon dioxidesupercritical fluid extract of adlay bran (B).

The term “C1 to C7 alcohol” as used herein refers to linear or branched,substituted or unsubstituted, mono- or poly-functional, and saturated orunsaturated alcohol; preferably unsubstituted, mono-functional andsaturated alcohol. In one preferred embodiment of the invention, the C1to C7 alcohol is selected from the group consisting of methanol,ethanol, n-propanol, isopropanol, n-butanol, iso-butanol, sec-butanol,tert-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol,2-methyl-2-butanol, 3-methyl-2-butanol, 3-methyl-1-butanol,2,2-dimethyl-1-propanol, 1-hexanol, 2,4-hexadiene-1-ol,2-methyl-cyclopentanol, cyclohexanol, 1-heptanol, 2-heptanol, orcycloheptyl alcohol. More preferably, the C1 to C7 alcohol is methanolor ethanol; most preferably, the C1 to C7 alcohol is ethanol. The C1 toC7 alcohol can be used solely or in combinations.

The C1 to C7 alcohol as used herein is preferably an alcohol solutionwith a concentration of from about 49% (v/v) to about 99.9% (v/v); morepreferably from about 75% (v/v) to about 99.9% (v/v); still morepreferably from about 90% (v/v) to about 98.0% (v/v).

As used herein, the term “C1 to C7 alcohol extract of adlay bran (A)”refers to extract obtained by extracting the adlay bran with the C1 toC7 alcohol. The manner of extracting a part of a seed with a solution iswell-known to artisans skilled in this field. In one preferredembodiment of the invention, the adlay bran is soaked in the alcoholsolution for extraction; more preferably, the adlay bran is soaked inthe alcohol solution and subjected to ultrasonic vibration extraction.

The temperature of extraction is preferably from about 10° C. to about100° C.; more preferably from about 15° C. to about 50° C.; still morepreferably from about 20° C. to about 40° C.

In one preferred embodiment of the invention, the C1 to C7 alcoholextract of adlay bran (A) is prepared according to a process comprising:

-   -   (a) providing adlay bran;    -   (b) cutting the adlay bran into small pieces; and    -   (c) extracting the small pieces in step (b) with the C1 to C7        alcohol to obtain an extract.

According to the process of the invention, prior to step (b), the adlaybran is preferably dried.

In one preferred embodiment of the invention, step (b) further comprisesblending the small pieces into powder. The manner of cutting and/orblending is well-known to artisans skilled in this field.

The ratio (w/v) of the adlay bran and the alcohol solution is notspecifically restricted, and can be about 1:1 to about 1:10; preferablyabout 1:3 to about 1:8; and most preferably about 1:5.

In one preferred embodiment of the invention, the extraction step (c)can be repeated, and the extract is collected by merging.

Preferably, the process further comprises (d) obtaining a liquidfraction from the extract, and a solid fraction is removed. The mannerof removing the solid fraction to obtain the liquid fraction iswell-known to artisans skilled in this field.

Preferably, the process further comprises a condensing step. The mannerof condensing is well-known to artisans skilled in this field, such asby a reduced-pressure condenser.

The carbon dioxide supercritical fluid extract of adlay bran (B)according to the present invention refers to an extract obtained byextracting adlay bran with carbon dioxide supercritical fluid. Thesupercritical fluid refers to a homogenous fluid state finally obtainedwhen the properties of gas and liquid get similar at a temperaturehigher than the critical temperature and a pressure higher than thecritical pressure. Similar to gas, the supercritical fluid hascompressibility, and similar to fluid, has the fluidity, and can be usedfor extraction; moreover, a commercial supercritical fluid extractionequipment is available, for example, NATEX, SEPAREX, UHDE and TAIWANSUPERCRITICAL TECHNOLOGY Co., Ltd. provide commercial supercriticalfluid extraction equipments, of which the specifications are generallyindicated by the capacity of the extraction tank, such as 500 g to 2000kg for selection, so that a suitable supercritical fluid extractionequipment can be selected according to requirements.

In one preferred embodiment of the invention, the carbon dioxidesupercritical fluid extract of adlay bran (B) is extracted at a pressureof from about 150 bar to about 500 bar; more preferably from about 200bar to about 400 bar; still more preferably from about 350 bar to about380 bar.

In one preferred embodiment of the invention, the carbon dioxidesupercritical fluid extract of adlay bran (B) is extracted at atemperature of from about 30° C. to about 80° C.; more preferably fromabout 40° C. to about 70° C.; still more preferably from about 50° C. toabout 60° C.

In one preferred embodiment of the invention, the carbon dioxidesupercritical fluid extract of adlay bran (B) is extracted with a fluxof carbon dioxide supercritical fluid of from about 20 kg/h to about 50kg/h; more preferably from about 30 kg/h to about 45 kg/h; still morepreferably from about 38 kg/h to about 40 kg/h.

In one preferred embodiment of the invention, the extraction time forthe carbon dioxide supercritical fluid extract of adlay bran (B) is fromabout 40 minutes to about 100 minutes; more preferably form about 50minutes to about 80 minutes.

In one preferred embodiment of the invention, the carbon dioxidesupercritical fluid extract of adlay bran (B) is extracted in theexistence of a co-solvent such as about 1% to about 10% of 95% ethanol.

Preferably, the carbon dioxide supercritical fluid extract furthercomprises a condensing step. The manner of condensing is well-known toartisans skilled in this field, such as by a reduced-pressure condenser.

According to the invention, the weight ratio of the C1 to C7 alcoholextract of adlay bran (A) to the carbon dioxide supercritical fluidextract of adlay bran (B) is from about 3:1 to about 1:4. In the range,the pharmaceutical effect in treating a skin and/or subcutaneous tissuedisease is very effective; preferably, the ratio is from about 3:2 toabout 1:2; more preferably, the ratio is about 3:2, about 3:1, about 1:2or about 1:4.

In one preferred embodiment of the invention, the extract of adlay branis subjected to a high performance liquid chromatography assay. Thesample is prepared as a concentration of 1 g/ml by using acetone as asolvent and filtrated with a 0.45 μm filter. Twenty-μL filtrated sampleis analyzed by Hitachi® analysis HPLC. The column is Reverse phase C18column (250×4.6 mm i.d.; YMC Co., INC), and the detector is photo-diodearray detector. The column temperature is 40° C. The chromatograms areextracted at 280 nm. The mobile phase uses Solution A: 5% acetic acid inwater; Solution B: 0.5% acetic acid in water/100% acetonitrile (1:1,v/v). The gradient elution program is shown in Table 1.

TABLE 1 Time (min) Flux (mL/min) Solution A(%) Solution B(%) 0 1.0 90 1010 1.0 85 15 20 1.0 84 16 35 1.0 83 17 50 1.0 79 21 55 1.0 79 21

The spectrogram of the C1 to C7 alcohol extract of adlay bran (A) isshown in FIG. 1; the spectrogram of the carbon dioxide supercriticalfluid extract of adlay bran (B) is shown in FIG. 2; and the spectrogramof the extract of adlay bran with the 1:4 weight ratio of the C1 to C7alcohol extract of adlay bran (A) to the carbon dioxide supercriticalfluid extract of adlay bran (B) is shown in FIG. 3. As shown in FIG. 3,the spectrogram obtained comprises peaks at retention time of about 12.5minute to about 13.5 minute, about 14 minute to about 15.5 minute, about15.5 minute to about 16.5 minute, about 21 minute to about 22.5 minute,and about 32 minute to about 35 minute.

The composition according to the invention is preferably apharmaceutical composition, food composition or a cosmetic composition.

The pharmaceutical composition according to the invention is preferablyadministered topically or systemically by any method known in the art,including, but not limited to, intramuscular, intradermal, intravenous,subcutaneous, intraperitoneal, intranasal, oral, mucosal or externalroutes. The appropriate route, formulation and administration schedulecan be determined by those skilled in the art. In the present invention,the pharmaceutical composition can be formulated in various ways,according to the corresponding route of administration, such as a liquidsolution, a suspension, an emulsion, a syrup, a tablet, a pill, acapsule, a sustained release formulation, a powder, a granule, anampoule, an injection, an infusion, a kit, an ointment, a lotion, aliniment, a cream or a combination thereof. If necessary, it may besterilized or mixed with any pharmaceutically acceptable carrier orexcipient, many of which are known to one of ordinary skill in the art;see paragraph [0037] for example.

The external route as used herein is also known as local administration,includes but is not limited to administration by insufflation andinhalation. Examples of various types of preparation for localadministration include ointments, lotions, creams, gels, foams,preparations for delivery by transdermal patches, powders, sprays,aerosols, capsules or cartridges for use in an inhaler or insufflator ordrops (e.g. eye or nose drops), solutions/suspensions for nebulisation,suppositories, pessaries, retention enemas and chewable or suckabletablets or pellets or liposome or microencapsulation preparations.

Ointments, creams and gels, may, for example, be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agent and/or solvents. Such bases may thus, for example, includewater and/or an oil such as liquid paraffin or a vegetable oil such asarachis oil or castor oil, or a solvent such as polyethylene glycol.Thickening agents and gelling agents which may be used according to thenature of the base include soft paraffin, aluminium stearate,cetostearyl alcohol, polyethylene glycols, woolfat, beeswax,carboxypolymethylene and cellulose derivatives, and/or glycerylmonostearate and/or non-ionic emulsifying agents.

Lotions may be formulated with an aqueous or oily base and will ingeneral also contain one or more emulsifying agents, stabilising agents,dispersing agents, suspending agents or thickening agents.

Powders for external application may be formed with the aid of anysuitable powder base, for example, talc, lactose or starch. Drops may beformulated with an aqueous or non-aqueous base also comprising one ormore dispersing agents, solubilising agents, suspending agents orpreservatives.

Spray compositions may for example be formulated as aqueous solutions orsuspensions or as aerosols delivered from pressurised packs, such as ametered dose inhaler, with the use of a suitable liquefied propellant.Aerosol compositions suitable for inhalation can be either a suspensionor a solution. The aerosol composition may optionally contain additionalformulation excipients well known in the art such as surfactants e.g.oleic acid or lecithin and cosolvents e.g. ethanol.

Topical preparations may be administered by one or more applications perday to the affected area over the skin areas occlusive dressings mayadvantageously be used. Continuous or prolonged delivery may be achievedby an adhesive reservoir system.

The cosmetic composition according to the invention may be an aqueousphase formulation consisting essentially of water; it may also comprisea mixture of water and of water-miscible solvent (miscibility in waterof greater than 50% by weight at 25° C.), for instance lowermonoalcohols containing from 1 to 5 carbon atoms such as ethanol orisopropanol, glycols containing from 2 to 8 carbon atoms, such aspropylene glycol, ethylene glycol, 1,3-butylene glycol or dipropyleneglycol, C3-C4 ketones and C2-C4 aldehydes, and glycerin. Such an aqueousformulation preferably is in a form of aqueous gel or hydrogelformulation. The hydrogel formulation comprises a thickening agent tothicken the liquid solution. Examples of the thickening agents include,but are not limited to, carbomers, cellulose base materials, gums,algin, agar, pectins, carrageenan, gelatin, mineral or modified mineralthickeners, polyethylene glycol and polyalcohols, polyacrylamide andother polymeric thickeners. The thickening agents which give thestability and optimal flow characteristics of the composition arepreferably used.

The cosmetic composition according to the present invention may be in aform of emulsion or cream formulation. It can contain emulsifyingsurfactants. These surfactants may be chosen from anionic and nonionicsurfactants. Reference may be made to the document “Encyclopedia ofChemical Technology, Kirk-Othmer”, volume 22, pp. 333-432, 3rd edition,1979, Wiley, for the definition of the properties and functions(emulsifying) of surfactants, in particular pp. 347-377 of saidreference, for the anionic and nonionic surfactants.

The surfactants preferably used in the cosmetic composition according tothe invention are chosen from: nonionic surfactants: fatty acids, fattyalcohols, polyethoxylated or polyglycerolated fatty alcohols such aspolyethoxylated stearyl or cetylstearyl alcohol, fatty acid esters ofsucrose, alkylglucose esters, in particular polyoxyethylenated fattyesters of C1-C6 alkyl glucose, and mixtures thereof; anionicsurfactants: C16-C30 fatty acids neutralized with amines, aqueousammonia or alkaline salts, and mixtures thereof. Surfactants which makeit possible to obtain an oil-in-water or wax-in-water emulsion arepreferably used.

The cosmetic composition according to the invention may further comprisean effective amount of a physiologically acceptable antioxidant selectedfrom the group consisting of butylated p-cresol, butylated hydroquinonemonomethyl ether, and a tocopherol.

The cosmetic composition according to the invention may further comprisenatural or modified amino acid, natural or modified sterol compound,natural or modified collagen, silk protein or soy protein.

The cosmetic composition according to the invention are preferablyformulated for topical application to keratin materials such as theskin, the hair, the eyelashes or the nails. They may be in anypresentation form normally used for this type of application, especiallyin the form of an aqueous or oily solution, an oil-in-water orwater-in-oil emulsion, a silicone emulsion, a microemulsion ornanoemulsion, an aqueous or oily gel or a liquid, pasty or solidanhydrous product.

The cosmetic composition according to the invention may be more or lessfluid and may have the appearance of a white or colored cream, anointment, a milk, a lotion, a serum, a paste, a mousse or a gel. It mayoptionally be topically applied onto the skin in the form of an aerosol,a patch or a powder. It may also be in solid form, for example, in theform of a stick. It may be used as care products and/or as makeupproducts for the skin. Alternatively, It may be formulated as shampoosor conditioners.

In known fashion, the cosmetic composition according to the inventionmay also contain additives and adjuvants that are common in cosmetics,such as hydrophilic or lipophilic gelling agents, preservatives,antioxidants, solvents, fragrances, fillers, pigments, odor absorbersand dyestuffs.

The extract of adlay bran can be added to a conventional foodcomposition (i.e. the edible food or drink or precursors thereof) in themanufacturing process of the food composition. Almost all foodcompositions can be supplemented with the extract of adlay bran of theinvention. The food compositions that can be supplemented with theextract of adlay bran of the invention include, but are not limited to,candies, baked goods, ice creams, dairy products, sweet and flavorsnacks, snack bars, meal replacement products, fast foods, soups,pastas, noodles, canned foods, frozen foods, dried foods, refrigeratedfoods, oils and fats, baby foods, or soft foods painted on breads, ormixtures thereof.

The invention also provides a method for treating a skin and/orsubcutaneous tissue disease in a subject, which comprises administeringto said subject an effective amount of extract of adlay bran andoptionally a pharmaceutically acceptable carrier or excipient, whereinthe extract of adlay bran comprises C1 to C7 alcohol extract of adlaybran (A) and carbon dioxide supercritical fluid extract of adlay bran(B), and the weight ratio of the C1 to C7 alcohol extract of adlay bran(A) to the carbon dioxide supercritical fluid extract of adlay bran (B)is from about 3:1 to about 1:4.

The invention also provides use of extract of adlay bran in themanufacture of a medicament of the treatment of a skin and/orsubcutaneous tissue disease, wherein the extract of adlay bran comprisesC1 to C7 alcohol extract of adlay bran (A) and carbon dioxidesupercritical fluid extract of adlay bran (B), and the weight ratio ofthe C1 to C7 alcohol extract of adlay bran (A) to the carbon dioxidesupercritical fluid extract of adlay bran (B) is from about 3:1 to about1:4.

The “the skin and/or subcutaneous tissue disease” as used herein refersto a disease occurring in the skin and/or subcutaneous tissue. In onepreferred embodiment of the invention, the skin and/or subcutaneoustissue disease is selected from the group consisting of inflammation,hair follicle damage, skin atrophy, bruising, burn, cheilitis, dry skin,flushing, alopecia, hyperpigmentation, hypopigmentation, induration,fibrosis, injection site reaction, extravasation change, nail change,photosensitivity, pruritus, itching, rash, desquamation, acne,acneiform, dermatitis associated with radiation, erythema multiforme,hand-foot skin reaction, skin breakdown, decubitus ulcer, striae,telangiectasia, ulceration, and urticaria.

In one preferred embodiment of the invention, the skin and/orsubcutaneous tissue disease is caused by radiation. Preferably, theradiation is radiation of the radiation therapy for treating a tumor.The tumor is preferably a breast tumor or a lung tumor.

The invention further provides a method for lowering interleukin-1α,interleukin-1β, interleukin-6, tumor necrosis factor-α, interleukin-8,prostaglandin-2 and/or C-Reactive Protein in a subject, which comprisesadministering to said subject an effective amount of extract of adlaybran and optionally a pharmaceutically acceptable carrier or excipient,wherein the extract of adlay bran comprises C1 to C7 alcohol extract ofadlay bran (A) and carbon dioxide supercritical fluid extract of adlaybran (B), and the weight ratio of the C1 to C7 alcohol extract of adlaybran (A) to the carbon dioxide supercritical fluid extract of adlay bran(B) is from about 3:1 to about 1:4.

The invention also provides use of extract of adlay bran in themanufacture of a medicament of lowering interleukin-1α, interleukin-1β,interleukin-6, tumor necrosis factor-α, interleukin-8, prostaglandin-2and/or C-Reactive Protein, wherein the extract of adlay bran comprisesC1 to C7 alcohol extract of adlay bran (A) and carbon dioxidesupercritical fluid extract of adlay bran (B), and the weight ratio ofthe C1 to C7 alcohol extract of adlay bran (A) to the carbon dioxidesupercritical fluid extract of adlay bran (B) is from about 3:1 to about1:4.

The following examples are given for the purpose of illustration onlyand are not intended to limit the scope of the present invention.

Example 1 Animal Model

Drugs and reagents: Adlay seeds (C. lachrymajobi L., Taichung Shuenyu no4, TCS4) are purchased from the farmers in Taichung County, Taiwan.

C1 to C7 alcohol extract of adlay bran (A): The shell and the seed coatof adlay are removed by using a mill, and adlay bran is milled intopowder, immersed in 3 folders of 95% ethanol (w/v) at 25° C., andsubjected to ultrasonic vibration extraction for 24 hours. Extracts ofthree times extraction are combined, and condensed at reduced pressureat 50° C., to obtain ethanol extract of adlay bran.

Carbon dioxide supercritical fluid extract of adlay bran (B): The shelland the seed coat of adlay are removed by using a mill, and adlay branis milled into powder and fed into a stainless steel extraction innertank, and then the inner tank is placed into an extraction tank ofsupercritical extraction equipment. The extraction conditions are:extraction pressure: 360 bar; extraction temperature: 55° C.; carbondioxide flux: 38 to 40 kg/h; co-solvent: 2% of 95% ethanol; extractiontime: 60 min. The extract is collected, and condensed by areduced-pressure condenser.

Extract weight ratio: The feeding dose of the adlay bran extract isselected to be 100 mg/Kg and 100 μL/day, and the tube feeding volume is100 μL/mouse. The adlay bran extract used in the experiments issuspended in 0.5% CMC (carboxymethyl cellulose) through ultrasonicvibration and is fed through a tube, and the mice are sacrificed at the35th day. The 25 types of different feeding doses are obtained bycombining the feeding dose of the C1 to C7 alcohol extract of adlay bran(A) being 0, 100, 200, 300 and 400 mg/kg and the feeding dose of thecarbon dioxide supercritical fluid extract of adlay bran (B) being 0,100, 200, 300, 400 mg/kg, and the tube feeding volume is 100μL/mouse/day.

Discussion on activity and mechanism of extract against side effect ofinflammation of the skin caused by radiation therapy of animal tumor:Adlay capsule is feed to animals through a tube (test group, extract A:extract B is 1:4), and the effect of different doses in alleviating theside effect of inflammation of the skin caused by radiation therapy istested. In the animal model, mouse breast cancer cells (4T1, 1×10⁶cells/mouse) are implanted into BALB/c mouse leg, when the tumordiameter is about 4 mm, different doses of test samples are fed througha tube, and two days later, radiation therapy (irradiation 5 Gy eachday, five day in total) is started and the adlay bran extract is fedthrough a tube every day, till the experiment is ended. In theexperiment process, the tumor size and the weight are measured and theblood is sampled every five days. The agent Sulfasil for treating theskin injuries in current clinical radiation therapy is used as thecontrol group. After the mice are sacrificed, tissue slices of the tumorradiation therapy part are made for epidermis and hair follicle tissuepathologic examination and determine the damage of the epidermis cellsand fibroblasts in the epidermis tissue by adopting TUNEL assay, incombination with pathology, and detects expression ofinflammation-related molecules such as NF-kB, COX-2, IL-1β and TNF-α byusing immunostaining. The slice results are shown in FIG. 4.

Results of weight change are shown in FIG. 5, the test samples areadministrated to the tumor animals when radiation therapy isadministrated, and no significant difference in weight change existsbetween groups; it can be known from FIG. 6 that, the test samples areadministrated to the tumor animals when radiation therapy isadministrated, the tumor volume is significantly reduced, compared withthe blank group, and no significant difference exists between the groupswith radiation therapy administrated.

In another aspect, adlay capsules (test group, extract A: extract B is1:4) of 200 and 400 mg/kg significantly reduce the skin inflammationcaused by radiation therapy, and at the same time, inhibit the increaseof serum IL-1α (FIG. 7), IL-1β (FIG. 8), IL-6 (FIG. 9), TNF-α (FIG. 10),IL-8 (FIG. 11), PGE2 (FIG. 12) and CRP (FIG. 13) caused by radiationtherapy; 100 mg/kg adlay capsule merely has significant inhibitioneffect on some indexes. It can be known from the results that, adlaycapsules of 200 and 400 mg/kg have significant effect in reducing furtissue injuries and inhibiting animal in vivo inflammatory factorindexes.

Alleviating the skin and/or subcutaneous diseases caused by radiationtherapy: The steps are performed as described in the safety evaluation,except that toxicity grading of in vivo epidermis response caused bytumor radiation therapy is evaluated every seven days in the studyprocess. In the in vivo epidermis response toxicity grading method, theCTCAE (Common Terminology Criteria for Adverse Events v3.0) system thatis most commonly used in toxicity evaluation in clinical treatment isadopted, and the grading is respectively performed through macroscopicobservation and microscopic pathological tissue biopsy in clinicalphysiology, and quantitative evaluation is performed by using a skinresponse score table. Pathological tissue slices of the treatment partare made. The results are shown in FIG. 14.

In quantization of the skin response, CompuSyn (for Drug Combinationsand for General Dose-Effect Analysis) software program is used tocalculate the combination of synergistic effect of experimentalmaterials in alleviating the skin responses and fur tissue injury causedby radiation therapy. The results are shown in Table 2:

TABLE 2 uantitative Extract (B) Extract (A) evaluation of furcombination (mg/kg) (mg/kg) tissue injury index 100.0 300.0 0.96 0.68150200.0 300.0 0.96 0.75281 400.0 100.0 0.96 0.48865 400.0 200.0 0.960.69204

As for the combination index (CI), according to definitions that CIvalue <1 is the addition effect, CI value=1 is the independent effect,and CI value >1 is the antagonistic effect, a combination of four bestCIs (CI<0.8) obtained through screening in the experiment are shown inTable 2, indicating significant inhibition on fur tissue inflammation,in which extract A:extract B is in the range of 3:1 to 1:4.

It can be known from the results in Table 2 and FIG. 14 that, in theanimal tumor radiation model in this embodiment, inflammation, hairfollicle damage, hair loss, radiation dermatitis and/or hand/foot skinresponses of the animals can be alleviated. Therefore, the extract ofadlay bran according to the present invention can be used to treat theskin and/or subcutaneous tissue diseases, especially inflammation, hairfollicle damage, hair loss, radiation dermatitis and/or hand/foot skinresponses.

Example 2 Human Experimentation of Alleviating Side Effects of RadiationTherapy on Breast Cancer Patients

Two adlay capsules (test group, extract A:extract B is 1:4) or olive oilcapsules (control group) (500 mg/capsule) are administrated afterbreakfast and dinner every day, and four capsules are administrated intotal each day.

In this clinical trial, prospective and random grouping and double blindmethod are adopted, a test group and a control group are designed, andthe clinical trail is carried out in two stages: At the first stage,response of common people to adlay capsule is first tested, in which twocapsules (500 mg/capsule) are administrated after breakfast and dinnerevery day, four capsules are administrated in total each day, and theadministration continues for four weeks. Blood examination is performedbefore test: including blood routine, liver function (including GOT andGPT), renal function (including BUN and CRTN), routine blood parameters(including cholesterol, HDL, LDL, TG and AC-sugar). After four-weekcontinuous administration, the same blood examination is performedagain, and after determining that no significant side effect exists, thesecond stage test is started: whether the adlay capsule can effectivelyreduce the skin responses caused by radiation when breast cancerpatients receive radiation therapy and whether the quality of life isimproved are determined; changes of immune function before and after thebreast cancer patients receive radiation therapy are determined throughblood examination, whether the adlay capsule can effectively improve theimmune function is determined, and statistical analysis is performed onthe clinical skin responses and physiological changes.

In the second stage test, breast cancer patients that meet the subjectconditions are divided into two groups: one group is the test group, andthe other group is the control group. For the test group, in the periodof radiation therapy, two adlay capsules (500 mg/capsule) areadministrated after breakfast and dinner every day, four capsules areadministrated each day, and the administration continues for 5 to 7weeks; for the control group, a substitute (placebo, the content is liveoil) with the same package is administrated in the same administrationmanner. The two groups are subjected to blood examination, the qualityof life questionnaire and physical examination of the skin before andafter treatment.

Number of Patients:

First stage: 10 common people

Second stage: 80 breast cancer patients in the test group and thecontrol group respectively.

-   -   170 subjects in total in the two stages.        Evaluation Method:

Safety test at the first stage: The subjects are subjected to bloodexamination for blood routine, liver function, renal function, androutine blood parameters, to determine whether serious abnormalresponses are caused due to administration of the adlay capsule. Thesecond stage: After a breast cancer patient agree to accept the test andsigns the subject consent form after explanation, the patient israndomly divided into the test group or the control group, and issubjected to blood examination: including blood routine, liver function(including GOT and GPT), renal function (including BUN and CRTN),cytokine (including IL-1, IL-6 and IL-8) and routine blood parameters(including cholesterol, HDL, LDL, TG and AC-sugar), quality of life andfatigue questionnaire, physical examination of the skin (including: thepH value, skin surface moisture and grease, moisturizing function,color, temperature, elasticity and skin Doppler ultrasound). Afterradiation therapy is started, the skin responses and physicalexamination of the patient in the period of treatment are recorded everyweek, including the skin response and changes in color in the chest walltreatment. After radiation therapy is ended, the patients are subjectedto blood examination and the quality of life questionnaire and physicalexamination of the skin the same as those before treatment.

Radiation Therapy:

Each breast cancer patient receives 5 to 7-week radiation therapy, andthe subject needs to take the adlay capsule or substitute orally everyday. In the radiation therapy, treatment range and volume are formulatedthrough computer tomography, and the dose distribution and doseuniformity are obtained through a computer treatment operation system,and the skin responses and other side effects are recorded upon revisitof the patient every week.

Results:

Blood examination is performed before and after administration of thetest product, the administration continues for four weeks, one patienthas abdominal distension, and no adverse events or serious side effectsexist. In the second stage test, 67 patients participate in subjectscreening, and 51 patients are enrolled, 16 patients among the subjectshave no intention to participate in this program because of: allergy,edema and discomfort of lower extremity caused by chemical treatment,being unable to cooperate the treatment due to busy work, no intention,having taking too much drugs and being unable to participate thetreatment, the rest patients sing the consent form, is subjected toblood examination, physical examination of the skin, photographicrecording of the affected part, questionnaire and administration of thetest product.

At the second stage, 2 patients have abdominal distension, and 1 patienthas loose stool, in which one patient stops participating the test dueto side effects such as abdominal distension, and one patient quits thetest. According to the treatment plan, the period of treatment for eachsubject is about 5 to 7 weeks, and 160 subjects are expected to beenrolled. In order to improve the intension of the subject ofparticipating this plan, increase the sample number, and increase thereliability of data collection, the ratio of the test group to thecontrol group is adjusted from 1:1 to 2:1. The number of individualcases of which the data is analyzable is 27, and the analytical resultsare shown in Table 3 to Table 6.

Table 3 shows comparison of average values of blood examination ofcommon people before and after administration of the adlay capsule atthe first stage, in which no difference before and after administrationexists between the test group and the control group, indicating that theadlay capsule will not affect the functions of liver and kidney.

TABLE 3 Average pretest(±SD) Average posttest(±SD) p-value Ages  54.9(11.36) Height 159.1 (4.31)  weight 58.3 (6.57) RBC  4.4 (0.65)  4.4(0.79) 0.648 hemoglobin 13.3 (1.30) 13.3 (1.09) 0.870 WBC  5.7 (1.22) 5.6 (0.83) 0.799 Hct 38.8 (3.45) 39.5 (3.06) 0.285 platelet 267.1(29.13) 265.7 (41.35) 0.859 BUN 11.2 (3.46) 12.0 (1.83) 0.375 creatinine 0.6 (0.10)  0.6 (0.13) 0.670 GOT 26.4 (5.85) 23.8 (5.27) 0.045 GPT 25.2(10.1) 22.5 (9.64) 0.158 cholesterol 195.8 (44.70) 201.4 (33.07) 0.523HDL  51.7 (13.84)  50.6 (11.39) 0.393 LDL 117.5 (34.46) 120.8 (25.63)0.688 TG 100.2 (52.45) 105.8 (64.24) 0.491 ACsugar 103.0 (24.21) 109.6(30.71) 0.024

Table 4 shows grading of the skin response RTOG/EORTC indexes whenbreast cancer patients receive treatment, and it can be seen from thepreliminary analysis of the results that, 3/14 (21.4%) of the patientsin the test group have a skin response grade greater than 1, and 6/13(46.2%) of the patients in the control group have a skin response gradegreater than 1.

TABLE 4 Experiment Control p- group(n = 14) group(n = 13) value Skin0.342 reaction Grade 0 4 (28.6%) 1 (7.7%)  Grade 1 7 (50.0%) 6 (46.2%)Grade 2 3 (21.4%) 5 (38.5%) Grade 3 0 (0.0%)  1 (7.7%)  Skin 0.173reaction ≦Grade 1 11 (78.6%)  7 (53.8%) >Grade 1 3 (21.4%) 6 (46.2%)Grade 0: No response Grade 1: with follicles pale, erythema, hair loss,reduced sweating and dry peeling Grade 2: with moderate erythema andpeeling and edema of patchy infiltration, more obvious at the skin foldsfor most patients Grade 3: with peeling and trauma of infiltration, andabrasion and bleeding Grade 4: with necrosis, ulcer, spontaneousbleeding, and other symptoms Grade 5: dead

Table 5 shows clinical and pathological features of breast cancerpatients after administration of the adlay capsule, in which nodifference in distribution exists between the test group and the controlgroup.

TABLE 5 Experiment group Control group p- (n = 14) (n = 13) value Ages51.4 (±13.7)    51.6 (±10.7)  0.969 clinical stage of primary 0.733tumor 0 1 (7.1%)  1 (7.7%) 1 3 (21.4%) 1 (7.7%) 1b 0 (0.0%)  1 (7.7%) 1c3 (21.4%)  5 (38.5%) 2 5 (35.7%)  4 (30.8%) 3 1 (7.1%)  0 (0.0%) is 1(7.1%)  1 (7.7%) clinical stage of primary 0.340 tumor 1a 3 (21.4%) 1(7.7%) 1b 3 (21.4%)  3 (23.1%) 1c 3 (21.4%)  5 (38.5%) 2 3 (21.4%)  2(14.3%) 3 2 (14.3%) 0 (0.0%) is 0 (0.0%)   2 (14.3%) Pathologicalclassification 0.308 Infiltrating ductal 13 (92.9%)  10 (76.9%)carcinoma Infiltrating lobular 1 (7.1%)  1 (7.7%) carcinoma Ductalcarcinoma in situ 0 (0.0%)   2 (15.4%) Surgical classification 0.511breast conserving surgery 12 (85.7%)  11 (84.6%) modified radical 0(0.0%)  1 (7.7%) mastectomy Others 2 (14.3%) 1 (7.7%) Radiation therapy0.363 conventional radiation 4 (28.6%)  4 (30.8%) therapy intensitymodulated 8 (57.1%)  9 (69.2%) radiation therapy tomotherapy 2 (14.3%) 0(0.0%)

Table 6 shows changes of blood examination before and after treatment ofbreast cancer patients after radiation therapy, in which no significantdifference exists.

TABLE 6 Experiment group (n = 14) Control group (n = 13) p-value RBC0.323 Pretest  4.12 (0.40) 4.04 (0.40) Posttest  4.37 (0.52) 4.13 (0.43)Hgb 0.310 Pretest 12.04 (1.72) 12.11 (1.55)  Posttest 12.54 (1.25) 12.20(0.94)  WBC 0.719 Pretest  5.24 (2.06) 5.74 (2.10) Posttest  3.79 (0.90)4.04 (0.89) Hct 0.219 Pretest 35.42 (4.45) 35.56 (4.14)  Posttest 37.18(3.17) 35.92 (2.54)  platelet 0.160 Pretest 231.60 (84.74) 273.54(68.67)  Posttest 172.00 (46.44) 179.15 (67.65)  BUN 0.179 Pretest 10.45(3.05) 11.86 (4.93)  Posttest 10.86 (3.59) 10.67 (3.95)  creatinine0.193 Pretest  0.57 (0.12) 0.62 (0.17) Posttest  0.59 (0.11) 0.61 (0.14)GOT 0.207 Pretest 25.86 (5.93) 22.85 (5.40)  Posttest 25.50 (4.45) 25.62(10.45) GPT 0.303 Pretest  24.14 (10.10) 22.92 (11.18) Posttest 22.93(6.74) 26.08 (15.75) Cho 0.584 Pretest 196.43 (35.72) 189.38 (29.11) Posttest 181.79 (35.71) 180.46 (24.44)  HDL 0.814 Pretest  46.30 (12.19)54.69 (14.73) Posttest  45.57 (10.80) 54.59 (11.03) LDL 0.593 Pretest121.26 (36.59) 54.69 (27.13) Posttest 111.35 (36.50) 97.13 (27.36) TG0.256 Pretest 122.14 (60.31) 100.69 (50.44)  Posttest 125.64 (51.51)161.62 (215.25) ACsugar 0.866 Pretest  99.64 (10.67) 103.62 (13.84) Posttest  97.93 (14.12) 101.38 (17.06) 

The results of this embodiment show that, dry skin, induration, rash,desquamation, acne, erythema multiforme, skin breakdown and/orulceration of the breast cancer patients can be alleviated; therefore,the extract of adlay bran according to the present invention can be usedto treat the skin and/or subcutaneous tissue diseases, especially dryskin, induration, rash, desquamation, acne, erythema multiforme, skinbreakdown and/or ulceration.

While embodiments of the present invention have been illustrated anddescribed, various modifications and improvements can be made by personsskilled in the art. It is intended that the present invention is notlimited to the particular forms as illustrated, and that all themodifications not departing from the spirit and scope of the presentinvention are within the scope as defined in the following claims.

What is claimed is:
 1. A pharmaceutical composition for treating skindisease in a human in need thereof consisting essentially of atherapeutically effective amount of a dehulled Coix lachryma-jobi L.seed extract which has been extracted with supercritical CO2 andethanol, wherein said pharmaceutical composition is in a form selectedfrom the group consisting of a tablet, a pill, a capsule, a transdermalpatch, a pessary, an ampoule, a retention enema bag, a pellet, and amicroencapsulation.